The overall goal of this competitive renewal is to identify pivotal mechanisms to regulate pulmonary macrophage gene expression that contribute to the molecular pathogenesis of lung inflammation in severe sepsis and are amenable to interdiction. Our published studies indicate that PU.1 is a critical regulator of mature macrophage function and that prophylactic conditional genetic ablation of PU.1 in mice greatly attenuates lung inflammation and permeability edema in response to endotoxemia. Our supportive data indicate that PU.1 regulates demethylation of Krppel-like factor 4 (KLF4) which allows the engagement of PU.1 to the KLF4 enhancer region resulting in transcriptional KLF4 gene expression. Ectopic over-expression of KLF-4 results in prolongation of NF-kB activation in macrophages and accentuates the duration of neutrophilic lung inflammation in mice suggesting that the pro-inflammatory effects of PU.1 are transduced through KLF4. Furthermore, we have shown that KLF4 and KLF2 may have antagonistic effects on the expression of the pro vs. counter-inflammatory macrophage inflammatory phenotype. We therefore hypothesize that pulmonary macrophages regulate the resolution of lung inflammation through epigenetic and transcriptional regulation of KLF4 gene expression by PU.1. In two specific aims that focus on whole animal models of acute lung injury we will determine whether PU.1 induction of KLF4 contributes to the pro-inflammatory (M1) macrophage phenotype whereas deficiency or competitive inhibition of KLF4 by KLF2 results in a phenotypic shift to a counter-inflammatory (M2) phenotype that is involved in restoring lung health. Specific Aim 1: To determine the necessity of PU.1 for KLF4 gene expression by pulmonary macrophages and determine the role of KLF4 in the pathogenesis of acute lung inflammation and injury. Specific Aim 2: To determine the impact of PU.1 on epigenetic regulation of KLF4 gene expression and the relative role of KLF4 and KLF2 in regulating the resolution of neutrophilic lung inflammation and injury. Our studies are designed to interrogate whether targeting of the influence of PU.1 on KLF4 gene expression is an effective treatment for acute lung inflammation and injury and could possibly be applied to other acute and chronic inflammatory conditions such as type 1 diabetes, inflammatory bowel disease, psoriasis, and rheumatoid arthritis. PUBLIC HEALTH RELEVANCE: The acute respiratory distress syndrome (ARDS) is a severe and life threatening condition that requires a mechanical breathing machine and other supportive care in an intensive care unit. We have shown that macrophages in the lung have an essential role in initiating the severe lung inflammation that causes ARDS. The purpose of this research is to define the basic molecular mechanisms that regulate macrophage involvement in ARDS in order to design new and effective treatments.